Syrups containing a mixture of glucose and fructose are widely used in industry because of their sweet taste and their low tendency to crystallize. Such syrups are commonly produced from glucose syrups using a glucose isomerase to catalyze the isomerization of glucose to fructose. Important for the economy of this process are low enzyme costs and negligible formation of by-products, that must be removed before the syrup can be used.
Glucose isomerases may be obtained from a large number of different species of microorganisms, and the properties of the glucose isomerases vary from species to species.
Once the microorganism for production of glucose isomerase has been selected it is of the utmost importance to carry out enzyme production in such a way that the enzyme cost is as low as possible. The enzyme cost is largely dependent upon the cost of the fermentor and upon the details of how the fermentation is carried out therein, notably the cost of the materials in the fermentation medium, the fermentation time, the power requirement, the concentration of enzyme in the fermentation broth and the yield of enzyme. In this connection it should be noted that production of many enzymes involved in catabolism of microorganisms e.g. glucose isomerase, are known to be reduced by presence of glucose and other energy rich compounds in the fermentation medium, the expression "repression" being used hereafter to identify the phenomenon of enzyme yield suppression by growth conditions. Thus, in order to get a better yield of glucose isomerase, other carbon sources than glucose could be used, e.g., glycerol, or alternatively the glucose content in the fermentation medium be controlled to growth-limiting concentrations.
Carbon limited growth can be carried out in three preferred ways. One is the dosed batch fermentation wherein a carbon and energy source is supplied to a batch culture in the post-log phase at a constant rate, which limits growth and relieves repression on enzyme synthesis. Another method is a fed batch fermentation (Pirt, S. J. (1974), J. appl. Chem. Biotechnol. 24, 415). A fed batch fermentation can be a homogeneous batch fermentation which is growth limited by the concentration of the carbon and energy source, where all other nutrients are present in excess. When the growth-limiting carbon and energy source is exhausted, the batch culture is supplied continuously with nutrient medium. Fermentation broth is removed discontinuously. The volume variation and the discontinuous removal of fermentation broth distinguishes the fed batch fermentation from a chemostat culture. Finally (and most efficiently) carbon limited growth can be carried out in the classical chemostat (Monod, J., A. Rev. Microbiol., 3, 64 (1949)). A chemostat culture consists of a perfectly mixed suspension of biomass into which medium is fed at a constant rate (F), and the culture is removed from the chemostat at the same rate, the volume of the culture (V) thus remains constant. In steady state the ratio F/V = D (dilution rate) is equal to the specific growth rate .mu.. Using this technique e.g. Smith and Dean (J. Gen. Microbiology 72, 37-47 (1972) found an increased yield of beta-galactosidase in lactose (the carbon and energy source) limited chemostat cultures of Klebsiella aerogenes.
During fermentation of glucose isomerase producing microorganisms, any growth-limitation due to oxygen is usually avoided. Thus normally a sufficient amount of air is supplied to the medium, or the fermentation is operated with growth-limiting factors other than oxygen, e.g. a growth-limitation due to a nutrient. Growth-limitation due to oxygen is unwanted because then carbon sources in excess will be repressing enzyme production.
Since the saturation concentration of oxygen in fermentation media is very low, conventional fermentors designed to the fermentation of glucose isomerase are constructed in a manner which provides a high transfer rate of oxygen from the gaseous phase to the liquid phase. Conventional fermentors for glucose isomerase fermentation thus are constructed with agitators designed to a high rotational velocity and/or with an efficient distributor means from the air intake and/or with an air intake with a high capacity of air blown into the fermentor per unit of time and/or with a fermentor design having a high ratio between height and width. The oxygen concentration in the fermentation medium might well be very low but still not be growth-limiting, due to the high transfer rate of oxygen from the gaseous phase to the liquid phase.
The concentration of oxygen in the fermentation medium is measured according to a polarographic or galvanic measuring method. The sensitivity of these methods is rather small; oxygen concentrations below 1.5 mm Hg can only be detected with difficulty in large scale operation.